It is well established that the hypothalamic gonadotropin-releasing hormone (GnRH) simulates an acute and augmented (GnRH priming) release of LH and that estradiol (E2) greatly enhances the LH response to GnRH. However, our knowledge of the cellular events involved in the biosynthesis and secretion of LH as regulated by GnRH and E2 is limited. The specific aims of this proposal are: 1) to determine if the augmented release of LH is dependent upon new synthesis of LH, how GnRH alters the biosynthesis of LH and how E2 alters the GnRH biosynthetic response; 2) to characterize the different molecular forms of immunoreactive LH in the anterior pituitary of female rats, if these forms are qualitatively or quantitatively altered by the in vivo and in vitro hormone environment, and if some forms are preferentially released; 3) to determine the influence of GnRH and E2 on the temporal pattern of synthesis of the various molecular forms of LH. Using the in vitro pituitary perifusion system as previously established by our laboratory, we will 1) determine the changes in total immunoreactive LH by radioimmunoassay (RIA) and the incorporation of radiolabeled amino acid (proline) and carbohydrate (glycosamine, mannose, galactose, fucose) into LH by immunoprecipitation at different times after GnRH stimulation and how this response is altered by previous exposure to E2; 2) determine the effect of the steroid hormone environment an the ability of GnRH to induce changes in the molecular forms of immunoreactive LH in the pituitary and that released from the gland. Differentiation of the molecular forms of LH will be assessed by lectin binding and gel isoelectric focusing techniques; and 3) determine the temporal synthesis and release of the different molecular forms of LH as a function of time of GnRH stimulation in pituitaries from ovariectomized rats and from those given physiological E2 replacement. The data obtained from the proposed experiments should collectively provide critical background information for future studies regarding the sites of action of GnRH and E2 on LH biosynthesis, and the biochemical mediators responsible for these events. Furthermore, the data could be used to more clearly formulate hypotheses as to how GnRH and E2 are involved in the regulation of LH secretion during the reproductive cycle. Understanding the basic physiological mechanism involved in this regulation would provide a better rationale for developing pharmacological methods to control these events in health and disease.